(a) Field of the Invention
The present invention relates to a method for the isolation from an aqueous medium of .alpha.-L-aspartyl-L-phenylalanine methyl ester (hereinafter abbreviated as .alpha.-APM) having a high rate of solution in water.
(b) Description of the Prior Art
.alpha.-APM is widely known as a dipeptide base sweetener. It has sweetness of good quality and a degree of sweetness about 200 times the sweetness of sucrose. The demand for .alpha.-APM as a diet sweetener is rapidly expanding.
.alpha.-APM is a dipeptide compound composed of L-aspartic acid and L-phenylalanine methyl ester. It can be prepared by both chemical and biochemical processes, the latter utilizing microorganisms. Various methods have been disclosed for each process.
As a typical example of a chemical process of producing .alpha.-APM, L-aspartic anhydride having a protected amino group is subjected to a condensation reaction with L-phenylalanine methyl ester in a suitable solvent and subsequently cleaving the protective group by a conventional method to obtain .alpha.-APM (For example, U.S. Pat. No. 3,786,039). In a representative biochemical process, N-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester are condensed in the presence of metalloprotease to obtain N-benzyloxycarbonyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester. The benzyloxycarbonyl group is then removed from the intermediate by catalytic reduction to give .alpha.-APM.
In the industrial production of .alpha.-APM, a step of purifying crude .alpha.-APM is inevitable for the preparation of final product by isolating .alpha.-APM from the reaction mass, whichever of the above mentioned processes is employed. The purification step is usually carried out by recrystallization from a solution of water or a water-containing solvent (water or a water-containing solvent is hereinafter referred to as aqueous medium). Another method for removing impurities involves a stirring treatment of a suspension of .alpha.-APM in an aqueous medium, depending upon the quality of crude .alpha.-APM. However, pure .alpha.-APM obtained by this purification method forms a hard block in the dry state. Therefore, crushing is required for the preparation of final product, which causes difficulties in the handling of .alpha.-APM. Also, a long period of time is required for the drying and the content of a diketopiperazine compound, which is an intramolecular cyclization product of .alpha.-APM, is liable to increase. Thus the method causes problems in the manufacture of product having uniform quality.
.alpha.-APM purified by conventional recrystallization methods has the disadvantage of poor solubilization (rate of solution) in water. For example, when .alpha.-APM obtained conventionally by recrystallization from a 50 vol. % aqueous methanol solution is crushed and 250 mg of the resultant powder is poured into 250 ml of water with stirring at the room temperature and the solubilization thereof is determined by alternatively and repeatedly stirring and standing every 30 seconds, a significant amount of undissolved .alpha.-APM remains even after 5 minutes and more than 15 minutes is required for it to dissolve completely. .alpha.-APM obtained by recrystallization from water also has similar low solubilization.
Because the present demand for .alpha.-APM as a sweetener is primarily in the field of soft drinks, the solubilization of .alpha.-APM in water is definitely an important factor in the determination of product specifications. However, there is little prior art relating to improving the solubilization of .alpha.-APM itself.
Japanese Laid-Open Patent No. 177952/1983 discloses a method for crystallizing .alpha.-APM from an aqueous solution in which the initial concentration of .alpha.-APM is 2-10 wt. % and the aqueous solution is cooled with control of heat conduction and without providing forced flow, such as with mechanical stirring. The total mass is converted to a pseudo-solid phase having the consistency of a sherbet and, if necessary, further cooled. The thus-produced crystals of .alpha.-APM have enhanced filterability and the improved properties of fine particles, such as bulk density and the like. The .alpha.-APM thus-obtained is described as having excellent solubilization as compared to conventionally crystallized product. Although this method certainly improves various properties of the fine particles of .alpha.-APM thus isolated, including solubilization to a remarkable extent, because the crystallization from the aqueous solution is carried out by cooling without forced flow, such as mechanical stirring, conventional crystallizing equipment requires a remarkably long time for the cooling process to complete. This requires an increase in equipment scale and restricts its industrial application. Therefore, the patent specifies the maximum distance between the cooling surface and cooling zone and proposes that a special crystallization device be employed to meet this requirement. Thus, as a practical matter, the method of Japanese Laid-Open Patent No. 177952/1983 cannot be employed industrially without using the special crystallization device described therein.
The present inventors have extensively investigated this solubilization problem in order to develop an isolation method for the preparation of easily soluble pure .alpha.-APM under stirred conditions using conventional equipment fitted with a usual stirrer. They discovered that if the isolation of .alpha.-APM from an aqueous medium was conducted in the presence of a certain additive, as a result, the resultant .alpha.-APM has a remarkably improved solubilization (dissolution rate) in water as compared to .alpha.-APM isolated conventionally in the absence of additive.